Establishment of a Three-Dimensional In Vitro Model of Taste Bud Cells for Fat Taste Investigation.

Fat taste is emerging as a sixth primary taste modality, with CD36 identified as a key receptor for oral detection of long-chain fatty acids. Tastants bind to receptors on taste bud cells (TBCs), triggering intracellular calcium mobilization, neurotransmitter release, activation of gustatory afferent fibers, and transmission of sensory information to the brain. Conventional two-dimensional (2D) culture systems fail to reproduce the complex three-dimensional (3D) architecture and cell-to-cell interactions essential in vivo. Here, we developed a 3D model of mouse TBCs to investigate the cellular mechanisms underlying fat taste perception. Immortalized CD36-positive TBCs and circumvallate papilla-derived TBCs were co-cultured under 2D and 3D conditions. In 3D conditions, TBCs self-organized into spheroids displaying organelles, intercellular junctions, and expression of markers of the three major TBC types. Compared to 2D-cultured cells, spheroids exhibited higher expression of taste receptors, including CD36, sweet receptor T1R2, umami receptor T1R1. Wnt/β-catenin and Sonic Hedgehog pathways, controlling differentiation, were upregulated. Spheroids showed enhanced responses to linoleic acid, with greater intracellular calcium mobilization and serotonin release. This model recapitulates more faithfully structural and functional properties of native taste tissue, providing a physiologically relevant in vitro platform for studies of fat taste mechanisms and potential factors modulating TBC function.